PROJECT SUMMARY It is also becoming clear that many diseases have an epigenetic component. However, in neurological diseases, such as neuropathic pain, it has been shown that certain genes for ion channels and pain receptors are downregulated and maintained through epigenetic silencing. Current treatments for neuropathic pain use pain killers which provide temporary relief for patients, but fail to cure damaged nerves. Further, the long-term use of pain killers carry a high risk of addiction. Thus, better approaches to treat neuropathic pain are urgently needed. Studies have linked changes in genes involve in neuropathic pain to the master transcriptional repressor, neuron- restrictive silencing factor (NRSF). NRSF silences gene expression by recruiting chromatin modifiers to leave repressive epigenetic marks including histone deacetylases, histone methytransferases, and DNA methytransferases, on neural genes involved in neuropathic pain. NRSF has been investigated as a therapeutic target and efforts to modulate its activity through small molecules, siRNA, and mimetics have been attempted. Since the transcriptionally repressive function of NRSF is mediated through epigenetic mechanisms, we propose that a Cas9-based approach to actively reverse repressive epigenetic marks left by NRSF on ion channel and pain receptor genes could provide a novel therapeutic strategy for treating neuropathic pain. This provides advantages over approaches using pan-acting small molecules and mimetics because it (1) would enzymatically remove and replace epigenetic marks, and (2) can be targeted to genes of interest in a sequence specific manner. To achieve these goals, first, we propose to develop an in vitro cell-based model of neuropathic pain dependent on NRSF-mediated repression, and secondly, we will explore a Cas9-based approach to reverse the repressive epigenetic marks brought about by NRSF on genes involved in neuropathic pain. This project will highlight epigenetic marks themselves as possible therapeutic targets and show that a Cas9-based system could be used therapeutically with a high degree of specificity.